1. Field of the Invention
The present invention relates to isolated and purified antigen which is expressed by a wild-type E. risticii strain and is specific to the strain. The present invention also relates to nucleic acid constructs which encode the antigen, expression vectors, transformed host cells, and methods for producing the antigen.
2. Discussion of the Background
Potomac horse fever (PHF), also known as equine monocytic ehrlichiosis (EME), is an acute infectious disease of horses. PHF was initially recognized in 1979 in areas along the Potomac river in Maryland and Virginia. The causative agent was subsequently identified in 1984 as Ehrlichia risticii, an obligatory intracellular rickettsial organism. Since then, PHF cases have been reported in many states of the U.S. and some provinces of Canada. Serological evidence suggests the presence of E. risticii in parts of Europe and Australia. The main disease features of PHF are fever, leukopenia, depression, anorexia and diarrhea. Some affected horses may also develop colic or laminitis. The mortality is as high as 20-25%. Recently, abortions in pregnant mares contracting the disease have been documented. PHF occurs mostly in the summer months. Although most of the rickettsial pathogens are transmitted by arthropod vectors and the seasonality of PHF also suggests this, all attempts to reveal the mode of transmission of E. risticii have been unsuccessful.
E. risticii infection is responsible for substantial economic loss to the equine industry. Currently, inactivated vaccines for PHF are commercially available from three different manufacturers. In endemic areas, vaccination of equine population against PHF is performed on a regular basis. Despite this, PHF is occurring in increasing numbers, including in vaccinated horses. In 1990, E. risticii was isolated from a horse suffering from severe PHF in spite of carrying a high titer of antibodies from multiple PHF vaccinations. On Western blot analysis, the antigenic profile of this newly isolated organism (90-12 strain) was considerably different from that of the original organism (25-D strain) isolated in 1984 during the initial outbreaks of the disease. In subsequent years, more isolates were obtained from vaccinated horses suffering from clinical PHF. These findings suggested the possible existence of strain variation in E. risticii and its probable role in vaccine failures in the field.
In the last few years, significant progress has been made toward understanding the pathogenesis and host immune response in E. risticii infection. Certain strains of mice have been identified to be good laboratory models of PHF. Various serological and DNA based tests have been developed to better facilitate diagnosis of the infection. Studies to identify the antigenic composition of the organisms and the major surface antigens involved in immune response were conducted. However, most of these studies have been performed with the original E. risticii isolates (isolated during 1984-85) from different laboratories. Except for one recent report on biological diversity in E. risticii isolates, no systematic comparison between different isolates has been made to identify the extent and importance of strain variation in this organism. Also, very little is known about the molecular biology of E. risticii. Hence, the present study has been undertaken to: i) understand the differences between the 25-D and 90-12 strains of E. risticii; ii) investigate the molecular basis of these differences; iii) identify protective antigen(s).
In addition to the main focus of problem solving E. risticii infections, there is an important scientific interest in these studies to gain more knowledge on ehrlichial organisms in general. Along with E. risticii, genus Ehrlichia of the family Rickettsiaceae contains some other recently identified organisms. New members of this genus include E. chaffeensis and E. ewingii, pathogens of human and dog, respectively. Recently identified human granulocytic ehrlichiosis (HGE) has been demonstrated to be caused by an organism similar to or the same as E. equi, an equine pathogen. Also, E. risticii has been found to infect dogs and cats. Emergence of these ehrlichial diseases and changes in host specificity of ehrlichial organisms are quite intriguing. Information on the important proteins of E. risticii and the genes they are encoded by may provide us with necessary clues to understand the sophisticated intracellular survival strategies of ehrlichial organisms and the natural dynamics in their ecosystem that lead to changes in their life cycles.
The present invention is based on the discovery that strains of Ehrlichia risticii express surface antigens that are specific to the strain. These surface-expressed proteins are termed strain-specific antigens (SSAs). These antigens have now been isolated and purified from the respective strains. The SSAs of the present invention may be used to detect Ehrlichia risticii strains and to generate a protective immune response against E. risticii strains, leading to the development of more effective vaccines against PHF.